Cryopreservation of Mouse Oocytes Using Sodium-Depleted Media: Comparison of HEPES-HTF and PBS-Based Formulations

Abstract

Background: Mammalian oocyte cryopreservation has gained considerable attention recently (Boldt, Human Reprod, 18:1250, 2003). Commercial products for cryopreservation of human oocytes by the traditional slow-cooling procedure are now available. To date, PBS-based sodium-depleted medium has been used to cryopreserve both mouse and human oocytes (Boldt, 2003; Stachecki et al, Biol Reprod, 59:395, 1998). Physiologically, HEPES-HTF-based medium would be preferred. Objective: Media based on HEPES-HTF and PBS were compared for their effectiveness to cryopreserve mouse oocytes. Materials and Methods: A 2x2 factorial experiment based on a modified HEPES-HTF and a PBS-based medium, with or without sodium-depleted formulations was performed. Freshly collected oocytes from superovulated females were pooled, divided into four equal groups (29-38/group) and cryopreserved in 0.5 mL straws using a standard slow-cooling procedure. Nine to 147 (av. 61) days later, the oocytes were thawed, inseminated in vitro and resulting embryos cultured to the blastocyst stage. The experiment was replicated four times with a total of 136 oocytes in each treatment. Recovery, survival, fertilization and development rates between the four treatments were analyzed by chi-square. Results: One third of all oocytes frozen in HEPES-HTF survived, were fertilized and developed to blastocysts. This was more than double the proportion in PBS-based media. The blastocyst development rates with the sodium-depleted HEPES-HTF based medium in the present study were 22% higher (39% v. 32%) than those reported by Stachecki et al (1998) using PBS-based sodium depleted medium. There were no significant differences between sodium-depleted and regular media in either of the two different formula based media. Eighty five percent of freshly collected control oocytes developed to blastocysts. Conclusions: A medium based on a modified HEPES-HTF formulation was superior to one based on PBS for the cryopreservation of mouse oocytes. This HEPES-HTF medium has been used for human oocytes with higher pregnancy rates than those obtained with PBS-based medium (Boldt, unpublished: Sage, data on file). The mouse model appears to emulate the results obtained in humans. The slow-cooling procedure used here can be readily adapted for cryopreserving human oocytes because most laboratories already have the equipment and experience with it to cryopreserve embryos.